Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy.

OBJECTIVE: To ascertain if hydrosalpinges are associated with reduced pregnancy rates and increased pregnancy loss after IVF-ET. Increased volume and leakage of hydrosalpinx fluid may exert negative effects on follicular development and embryo quality and/or render the uterine environment hostile to embryogenesis. We undertook this study to examine the effect of hydrosalpinx fluid on murine embryogenesis in vitro. DESIGN: Descriptive study. SETTING: Tertiary care facility. PATIENT(S): Premenopausal females undergoing salpingectomy or salpingostomy for hydrosalpinges. INTERVENTION(S): Collection of discarded hydrosalpinx fluid and development of a dose response curve for the effect of hydrosalpinx fluid on murine embryogenesis. MAIN OUTCOME MEASURE(S): Development of single cell mouse embryos in vitro. RESULT(S): All samples of tubal fluid obtained from hydrosalpinges demonstrated a significant embryo toxic effect at either the 100% or 10% concentration. Hydrosalpinx fluid demonstrated pH values (8.45 to 8.65) significantly higher than the physiologic range. Correction of pH to that of media did not affect cavitation rate. CONCLUSION(S): There is a well-defined and significant toxic effect of hydrosalpinx fluid. Procedures such as salpingectomy or proximal tubal occlusion to circumvent the passage of hydrosalpinx fluid into the uterine cavity may have beneficial effects on the developmental environment for embryos in vivo.

Correlations between periovulatory serum and follicular fluid CA-125 and granulosa cell hormones after controlled ovarian hyperstimulation.

OBJECTIVE: To determine the presence and concentration of CA-125 in periovulatory follicular fluid (FF) and serum after controlled ovarian hyperstimulation and to determine if the CA-125 in these two compartments could be related to granulosa cell markers such as inhibin or estradiol. STUDY DESIGN: Fifteen women undergoing controlled ovarian hyperstimulation for in-vitro fertilization-embryo transfer were studied. A transvaginal, ultrasound-guided follicular aspiration was performed. CA-125, inhibin, estradiol and FSH were measured in FF and serum. Pearson and Spearman's Rank Correlation tests were performed. RESULTS: CA-125 was measurable in 59% of follicles. Values ranged from undetectable to 3630 U/ml. Serum CA-125 ranged from undetectable to 126 U/ml. CA-125 and inhibin correlated negatively in FF and positively in serum. CONCLUSION: CA-125 was present in significant but variable concentrations in 59% of periovulatory follicles. A negative correlation was noted between CA-125 and inhibin or estradiol in the FF and a positive correlation with serum inhibin. No correlations were noted to oocyte retrieval or fertilization.

Comparative study of the survival rate of frozen-thawed embryos with perforated zonae from microsurgical fertilization and frozen-thawed embryos with intact zonae.

OBJECTIVE: To compare the survival rate of frozen-thawed embryos with perforated zonae from microsurgical fertilization (subzonal insemination) (SUZI) with that of embryos with intact zonae. DESIGN: Thirty-eight embryos resulting from microsurgical fertilization by SUZI were cryopreserved in 16 patient cycles. Within the same period, 140 zonae-intact embryos from 46 patient cycles were cryopreserved. The survival rate of the SUZI embryos was compared with the zonae-intact embryos after thawing. Clinical pregnancies were compared after the transfer of the thawed embryos. RESULTS: The total survival rates were 94.7% and 89.3% for thawed SUZI embryos and zonae-intact embryos, respectively. Within each type of embryo, total survival rates were similar irrespective of the age of the embryos at freezing. The blastomere loss per thawed embryo was the same for zonae-intact and SUZI embryos. One clinical pregnancy was obtained in patients who received thawed SUZI embryos and nine in patients who received embryos with intact zonae. CONCLUSIONS: It is concluded that SUZI embryos can be cryopreserved and thawed with the same degree of confidence as zonae-intact embryos. The low implantation rate of thawed SUZI embryos requires confirmation in a larger clinical series.

The effect of sperm preparation methods on the fertilization rate of oocytes micro-inseminated by subzonal sperm injection.

Three methods were used to prepare spermatozoa for subzonal injection into mature oocytes. In method A, the washed sperm suspension was incubated for 18 h in modified T6 culture medium. Method B consisted of incubating the sperm suspension for 6 h in regular T6 culture medium. In method C, the sperm suspension was incubated for 6 h in regular T6 culture medium containing 20% (v/v) follicular fluid. The percentages of acrosome-free spermatozoa and fertilization rates were compared for 42 treatment cycles assigned randomly to the three sperm preparation methods. The sperm suspensions prepared by methods A and C each had significantly higher proportions of acrosome-free spermatozoa compared to suspensions prepared by method B. The fertilization rates of oocytes micro-injected with spermatozoa prepared by methods A and C were significantly higher than for method B. Eight clinical pregnancies resulted from 28 cycles in which embryo replacement occurred. We conclude that the fertilization rate following subzonal sperm injection is related directly to the percentage of acrosome-free spermatozoa in the sperm suspension used for microinjection.

Species differences in susceptibility to 1,3-dinitrobenzene-induced testicular toxicity and methemoglobinemia.

The testicular toxicity and methemoglobinemia induced by 1,3-dinitrobenzene (1,3-DNB) was compared in two species, the Sprague-Dawley rat and the golden Syrian hamster. A marked difference in susceptibility to both endpoints of toxicity was observed. The hamster showed no testicular lesions at dose levels up to 50 mg/kg whereas, as previously reported by others, damage to rat testicular tubules in later stages of spermatogenesis was readily apparent at a 25 mg/kg dose level. Similarly, administration of 1,3-DNB induced substantially less methemoglobinemia in the hamster than in the rat. For example, at the 25 mg/kg dose level peak levels of methemoglobin in the hamster were 15% compared with 80% in the rat. Mortality in the rat also occurred at lower doses than in the hamster (50 vs 100 mg/kg, respectively). In in vitro studies, the capacity of 1,3-DNB and 1,3-DNB metabolites (nitroaniline, nitroacetanilide, aminoacetanilide, diacetamidobenzene) to induce methemoglobinemia was examined in suspensions of red blood cells obtained from both species. Only 1,3-DNB caused the formation of methemoglobin and rat red blood cells were twice as sensitive as hamster red blood cells. The species difference in susceptibility to both methemoglobinemia and testicular toxicity could indicate differences in 1,3-DNB clearance and/or formation of toxic metabolites. Additional metabolic work is under way. This study demonstrates that the hamster is more resistant than the rat to the testicular lesion and methemoglobinemia induced by 1,3-DNB.

Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons.

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport--phlorizin, which inhibits brush border (apical) Na+/glucose symporters, and ouabain, which inhibits Na+/K+-ATPase, thereby modifying the transmembrane Na+ gradient--were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain (L.M. Wiley, 1984, Dev. Biol. 105, 330-342) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10(-5) M; phlorizin, 10(-6) M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10(-4) M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.

A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice.

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres.

Cell surface and cytoplasmic polarity is exhibited by the blastomeres of mouse preimplantation embryos following compaction at the 8-cell stage of cleavage. It has been hypothesized that cytoplasmic polarity is initiated by plasma membrane functions of polar blastomeres that are absent from apolar blastomeres. To test this hypothesis the plasma membranes of "test" polar and apolar 8-cell- and 16-cell-stage blastomeres were inserted into the plasma membrane of "carrier" 4-cell-stage blastomeres by polyethylene glycol-mediated fusion of carrier-test blastomere pairs. After a 4-hr culture period each heterokaryon was scored for the distribution of two marker organelles--lipid droplets and nuclei--with respect to their proximity to the plasma membrane insert from the test blastomere. Plasma membrane inserts from polar test blastomeres were identified by labeling their apical domains with fluorescently tagged (succinylated) concanavalin A. The incidence of polar heterokaryons (those exhibiting a discrete fluorescently labeled area of plasma membrane corresponding to the apical domain inherited from the test blastomere) was 55/85 (69%) and 48/79 (61%) for 8-cell-stage and 16-cell-stage test blastomeres, respectively. In all polar heterokaryons, both nuclei were subjacent to the fluorescent label (apical domain of a polar plasma membrane insert), while the majority of lipid droplets resided in the hemisphere opposite the fluorescent label. In all 61 apolar heterokaryons examined (those lacking a discrete fluorescently labeled plasma membrane area) both nuclei were centrally located and lipid droplets were randomly distributed. These observations are consistent with the hypothesis that cytoplasmic polarity can be initiated by properties that distinguish the plasma membranes of polar blastomeres from those of apolar blastomeres.

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

Reproductive effects of chlorpromazine exposure to female mice: cell proliferation disadvantage revealed by the Chimera Embryo Assay.

Administration of chlorpromazine-HCl at 5 to 15 mg/kg bodyweight to pregnant CD-1 mice at 24 h after human chorionic gonadotropin (hCG) (20-23 h after mating) inhibited blastocyst formation and reduced the cell number of embryos recovered at 95 h after hCG. When embryos are recovered at the two- to four-cell stage (48-50 h after hCG) and cultured for an additional 47 h (to 95 h after hCG) or 72 h (to 120 h after hCG), blastocyst formation and embryo cell number were similarly reduced. When the dose range was reduced to 0.5 to 2 mg/kg bodyweight, no significant effect of the drug was observed on blastocyst formation or on embryo cell number. However, when aggregation chimeras were formed between embryos recovered from drug-exposed females and from untreated females, a decrease in cell proliferation rate of the embryo from the drug-exposed female was observed at a dose of 2 mg/kg bodyweight. This result indicates that exposing pregnant mice to chlorpromazine-HCl at doses as low as 2 mg/kg bodyweight can induce a potential for decreased cleavage rate in their pre-implantation embryos that can be revealed by challenging those embryos by direct contact with embryos from nonexposed females. Finally, when four-cell stage embryos recovered from untreated females cultured in the presence of chlorpromazine (0.1-25 mM), blastocyst formation and embryo cell number were significantly reduced in a dose-dependent manner. This last result suggests that in vivo the drug may act directly on the embryo from the pronuclear stage to the early morula stage of development.

Immunofluorescent localization of immunoglobulins on the cell surface of mouse oocytes and preimplantation embryos.

Living mouse oocytes and preimplantation embryos were assayed by indirect immunofluorescence for their ability to adsorb heterologous serum proteins from culture media to their cell surfaces. Bovine and human immunoglobulins of the IgG class were adsorbed by the oocytes and all stages of preimplantation embryos, while IgG of mouse or goat origin was not. In contrast, none of the serum albumins was adsorbed to the cell surfaces of mouse oocytes and preimplantation embryos. From the differential binding of IgG of some, but not all, of the species that were tested, we concluded that these cell surface IgG "receptors" on mouse oocytes and preimplantation embryos are likely to be heterophilic in nature. Similar observations were made irrespective of the strain of mice used to provide the oocytes and embryos. These observations raise the question of whether human oocytes and preimplantation embryos should also be assayed for their ability to adsorb animal serum proteins that are possible candidates as a substitute protein supplement for human serum in culture medium used in human in vitro fertilization/embryo transfer programs.

Ctenocephalides canis infestation of sheep and goats.

An investigation into infestation of sheep and goats with Ctenocephalides canis showed that while only a light degree of infestation was observed in the affected goats, light to heavy degrees of infestation occurred in sheep. In both species a light degree of infestation had no marked effect on the packed cell volume (PCV). Both medium and heavy degrees of infestation resulted in a significant lowering of the PCV of the affected sheep.